PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) is a clonal hematologic disorder caused by acquired somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene. The product of this gene takes part in the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor, a molecule that attaches numerous proteins to the cell membrane. Because PIG-A gene mutation originates in a multipotent hematopoietic stem cell with differentiation and self-renewal capacity, all progenitor cells deriving from the mutated HSC harbor the GPI defect, and have a complete or partial deficiency in the expression of GPI-anchored proteins (GPI-APs) on the cell surface. Some of these GPI-anchored proteins, such as CD55 and CD59, are regulators of the complement cascade, by interfering with the assembly of the terminal complement complex. In this manner, red blood cells of normal individuals are protected from complement-mediated destruction, whereas those deficient in anchored proteins such as CD55 and CD59 undergo hemolysis, predominantly intravascular. PNH is commonly associated with states of acquired bone marrow failure such as idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS). In AA, up to 60% of patients harbor small to moderate PNH populations, whereas in MDS, the prevalence of PNH clones is lower, 10-20%. Evaluation of deficient expression of GPI-APs in PB cells by flow cytometry (FC) is currently the method of choice for the laboratory diagnosis of PNH. Although most flow cytometric analyses have focused on testing the expression of the two GPI-APs CD55 and CD59, a fluorescently labeled inactive variant of the protein aerolysin (FLAER) emerged as a superior method for PNH testing in granulocytes and monocytes. PB red and white cells have been extensively studied in this disorder but there have only been few efforts to delineate in detail the abnormalities of BM cells in these patients. BM specimens are generally considered less suitable than PB owing to variable expression of GPI-APs during the various stages of hematopoietic cell development and are seldom studied for PNH evaluation. However, BM aspirates from patients with unexplained cytopenias, including bone marrow failure syndromes, are frequently submitted to laboratories for general diagnostic purposes but targeted PNH analysis is seldom performed on these samples. Specific goals of this project: 1. Analysis of fluorescently labeled aerolysin (FLAER) binding to bone marrow (BM) cells in normal volunteers and patients with primary marrow disorders such as aplastic anemia (AA) or myelodysplastic syndrome (MDS) who have detectable PNH in the peripheral blood (PB). 2. Comparison of PNH clone size in BM and PB. 3. Detection of PNH in BM by antibodies routinely used for diagnosis and characterization of BM disorders in clinical laboratories. 4. Assessment of proliferation and apoptosis of PNH and normal cell populations in the same BM samples. Methods: Flow cytometry analysis of FLAER binding, and expression of CD55/CD59 inn conjunction with other antibodies in BM cells. Proliferation and apoptosis assays by flow cytometry. Preliminary Results: FLAER binds to all normal BM cells, except for erythrocytes. FLAER binding increases with cell maturation and reaches the highest level on mature elements. In PNH, the clone size is smaller in lymphocytes than in other BM cells. PNH clone sizes in BM and PB are comparable, making BM equally suitable for PNH detection and quantitation. With antibodies routinely used for general diagnosis (anti CD45, CD13, CD11b, CD64 and the anchored proteins CD16 and CD14) PNH clones can also be detected in BM mature neutrophils and monocytes with high specificity and sensitivity. In rapidly expanding PNH populations, there appears to be an increase in proliferative fractions, in both red cells and neutrophils. Data continue to be analyzed currently. PRESENTATIONS 1. Dulau Florea A, Young NS, Maric I, Braylan RC. Human Pathology (Poster Abstract). Expression of Glycosylphosphatidylinositol (GPI) Anchor Protein (AP) in Bone Marrows of Normal Subjects and Aplastic Anemia Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH).Human Pathology (Poster abstract).The USCAP Annual Meeting 2016, Seattle, WA (March 12-18, 2016). 2. Dulau Florea A, Young NS, Maric I, Jordan EK, Jiang C, Ahmad F, Braylan R. Immunophenotypic Abnormalities Highly Suggestive of Paroxysmal Nocturnal Hemoglobinuria (PNH) can be Detected with Routine Flow Cytometric Analysis of Bone Marrow (BM). Human Pathology (Poster abstract).The USCAP Annual Meeting 2017, San Antonio, TX (March 4-10, 2017). 3. Dulau Florea A, Young NS, Jordan E, Maric I, Braylan R. 154 Bone Marrow Aspirate Samples are Equal to Peripheral Blood for the Detection of Paroxysmal Nocturnal Hemoglobinuria (PNH). Leuk Res. April 2017, Vol.55: S96 (Poster abstract). The 14th International Symposium on Myelodysplastic Syndromes (MDS 2017), Valencia, Spain (May 3-6, 2017).